Electrostatic Interaction between Oxysterol-binding Proteinand VAMP-associated Protein A Revealed by NMR andMutagenesis Studies*□S

Received for publication, November 5, 2009, and in revised form, February 22, 2010 Published, JBC Papers in Press, February 23, 2010, DOI 10.1074/jbc.M109.082602

Kyoko Furuita‡, JunGoo Jee‡§, Harumi Fukada¶, Masaki Mishima‡�, and Chojiro Kojima‡1

From the ‡Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, §Center for PriorityAreas, Tokyo Metropolitan University, Tokyo 192-0397, ¶Graduate School of Life and Environmental Sciences, Osaka PrefectureUniversity, Osaka 599-8531, and �Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan

Oxysterol-binding protein (OSBP), a cytosolic receptor ofcholesterol and oxysterols, is recruited to the endoplasmic retic-ulum by binding to the cytoplasmic major sperm protein (MSP)domain of integral endoplasmic reticulum protein VAMP-asso-ciated protein-A (VAP-A), a process essential for the stimula-tion of sphingomyelin synthesis by 25-hydroxycholesterol. Todelineate the interaction mechanism between VAP-A andOSBP, we determined the complex structure between theVAP-AMSP domain (VAP-AMSP) and the OSBP fragment con-taining a VAP-A binding motif FFAT (OSBPF) by NMR. Thissolution structure explained that five of six conserved residuesin the FFAT motif are required for the stable complex forma-tion, and three of five, including three critical intermolecularelectrostatic interactions, were not explained before. By com-bining NMR relaxation and titration, isothermal titration calo-rimetry, andmutagenesis experiments with structural informa-tion, we further elucidated the detailed roles of the FFATmotifandunderlyingmotions ofVAP-AMSP,OSBPF, and the complex.Our results show that OSBPF is disordered in the free state, andVAP-AMSP and OSBPF form a final complex by means of inter-mediates, where electrostatic interactions through acidic resi-dues, including an acid patch preceding the FFAT motif, prob-ably play a collective role. Additionally, we report that themutation that causes the familial motor neuron diseasedecreases the stability of the MSP domain.

Lipids are amajor component of cell membranes and also actas signaling factors. A variety of cytosolic lipid-binding proteinsis involved in lipid signal transduction and lipid transport (1).Because the endoplasmic reticulum (ER)2 membrane is a major

site for lipid biosyntheses,many cytosolic lipid binding proteinslocalize at the ER membrane to perform their functions. Oxys-terol-binding protein (OSBP) is a cytosolic receptor of choles-terol and oxysterols, such as 25-hydroxycholesterol, and hasbeen implicated to play a role in vesicle transport, lipid metab-olism, and signal transduction (2, 3). Wyles et al. (4) showedthatOSBP localizes at the cytosolic surface of the ERmembraneby interaction with integral ER protein VAMP-associated pro-tein (VAP-A) and identified the region of OSBP required forbinding to VAP-A. Shortly thereafter, Loewen et al. (5) foundthat the sequence EFFDAXE is conserved in OSBP and manyother lipid binding proteins and is a transcriptional regulatorspecific to phospholipid synthesis and showed that thesequence acts as an ER-targeting determinant by interactionwith VAP. Most of EFFDAXE sequences or sequences closelyrelated to EFFDAXE have acidic flanking regions (5); therefore,these sequences are referred to as FFAT motifs because theyconsist of two phenylalanines (FF) in an acidic tract (AT). It hasnow been shown that FFAT motifs in some lipid-binding pro-teins play a role in targeting to the ER (6–8).VAPs are type IImembrane proteins that are conserved from

yeast to human. Three VAP isoforms have been identified inhumans: VAP-A, VAP-B, and VAP-C (a splicing variant ofVAP-B) (9). VAPs generally localize at the ER, although theycan localize at other subcellular organelles in some species andcell types (10–15). Most VAPs are composed of three domains;amajor spermprotein (MSP) domain, a coiled-coil domain, anda transmembrane (TM) domain (Fig. 1A). The N-terminalregion contains the highly conserved MSP domain. VAPs bindto FFAT motifs by the MSP domain. VAPs are involved in adiverse range of cellular events. Before discovery of the FFATmotif, VAPs were mainly considered to play a role in vesicletrafficking given that VAPs were shown to interact with pro-teins involved in vesicular fusion, although precise details oftheir functions were unclear (12, 16–19). After discovery of theFFATmotif, VAPs were shown to have important roles in non-vesicular lipid transport (8), lipid metabolism (20), the regula-tion of ER structure (7, 21), and the unfolded protein response(14) through interaction with FFAT motifs. Additionally, theP56S mutation in human VAP-B causes autosomal dominant

* This work was supported by grants from the Ministry of Education, Culture,Sports and Technology (Japan) through Target Proteins Research Pro-gram, Global Centers of Excellence Program, and grants-in-aid for scientificresearch.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S7.

The atomic coordinates and structure factors (code 2rr3) have been deposited inthe Protein Data Bank, Research Collaboratory for Structural Bioinformatics,Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence should be addressed: Institute for ProteinResearch, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871,Japan. Tel.: 81-6-6879-8598; Fax: 81-6-6879-8599; E-mail: kojima@protein.osaka-u. ac.jp.

2 The abbreviations used are: ER, endoplasmic reticulum; OSBP, oxysterol-binding protein; ORP, OSBP-related protein; VAP-A, VAMP-associated pro-

tein; MSP, major sperm protein; ALS8, amyotrophic lateral sclerosis 8; ITC,isothermal titration calorimetry; DSC, differential scanning calorimetry;HSQC, heteronuclear single quantum correlation; DTT, dithiothreitol; WT,wild type; NOESY, nuclear Overhauser effect (NOE) spectroscopy.

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motoneuronal diseases and familial amyotrophic lateral sclero-sis 8 (ALS8), and P56S mutants of human VAP-B form aggre-gations in cells (22, 23). VAPs are important proteins whosefunctions are mediated by interaction of MSP domains withFFAT motifs.OSBP, which was the first lipid binding protein shown to

interact with VAP-A, requires interaction of the FFAT motifwith VAP-A to perform its function. In addition to the FFATmotif, OSBP has an N-terminal pleckstrin homology domainand a C-terminal lipid binding domain (Fig. 1A). OSBP usuallylocalizes in a vesicular compartment and the cytosol and trans-locates to the Golgi apparatus surface in response to increasesin cellular 25-hydroxycholesterol or depletion of cellular cho-lesterol (24). 25-Hydroxycholesterol stimulates sphingomyelinsynthesis (25), and the interaction between the FFAT motif ofOSBP and VAP-A is essential for stimulation of sphingomyelinsynthesis by 25-hydroxycholesterol (26). Proteins displayinghomology to the C-terminal lipid binding domain of OSBP arereferred to as OSBP-related proteins (ORPs) (2, 27). Humanspossess 12 ORP genes including OSBP (28). Four of the 12human ORPs contain the EFFDAXE sequence, and 4 containsequences closely related to EFFDAXE. In addition to these 8human ORPs, human lipid binding proteins containing FFATmotifs (Fig. 1B) have been investigated with respect to theirinteraction with VAPs and subcellular localization (Table 1).These results suggest that the EFFDAXE sequence alone is notsufficient for strong binding to VAP, and crucial residues maybe present in regions other than the EFFDAXE sequence. Itshould be noted that details of the interaction between OSBPand VAP-A remain unknown.In this study we have determined the solution structure of

the complex between the human VAP-A MSP domain (VAP-AMSP) and human OSBP fragment containing the FFAT motif

(OBSPF) in an effort to delineate the interaction betweenVAP-A and OSBP. The detailed binding mechanism was fur-ther investigated by NMR and isothermal titration calorimetry(ITC) combinedwithmutagenesis.We have successfully exam-ined 1) the stoichiometry between VAP-AMSP and OSBPF and2) the disorder property of OSBPF and 3) identified residueswithin and at the N-terminal side of the FFAT motif that con-tribute to the binding to VAP-AMSP. Additionally, we haveinvestigated the effect of the mutation that causes the diseaseALS8 using NMR and differential scanning calorimetry (DSC).

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The expression andpurification of unlabeled 13C,15N-labeled and 15N-labeledVAP-AMSP (human VAP-A E5-E128) and C-terminal hexahis-tidine-taggedOSBPF (humanOSBPG346-S379) with anN-ter-minal tryptophanwere as previously reported (32). Briefly, bothVAP-AMSP and OSBPF were overexpressed in Escherichia coliRosetta (DE3). Cells were lysed by sonication and centrifuged,and the supernatant was loaded onto a glutathione-Sepharose4B resin (GE Healthcare). The column eluate was then appliedto a Superdex 75 (26/60) gel filtration column (GEHealthcare).The fractions obtained were concentrated, and the glutathioneS-transferase tag was cleaved using PreScission Protease (GEHealthcare). Finally, the sample was applied to a Superdex 75(26/60) gel filtration column. ForOSBPF, nickel-Sepharose (GEHealthcare) was used in lieu of the aforementioned final gel-filtration step, and protein was eluted with a buffer containing300 mM imidazole.

Expression vectors for the site-directed mutants of OSBPF(E356K, E358A, F360A, E360Q, D361A, E364K, and E356K/E364K)were prepared usingQuikChange (Stratagene). The fol-lowing primers were designed to introduce the mutations:E356K forward (5�-gcgatgaagatgataagaatgaattttttg-3�) andreverse (5�-caaaaaattcattcttatcatcttcatcgc-3�); E358A forward(5�-agatgatgagaatgcattttttgatgcac-3�) and reverse (5�-gtgcatca-

FIGURE 1. A, domain structures of human VAP-A and human OSBP are shown.MSP, major sperm protein; CC, coiled-coil; TM, transmembrane; PH, pleckstrinhomology; LB, lipid binding. B, alignment of FFAT motifs of human lipid bind-ing proteins and rat ORP1 is shown.

TABLE 1Summary of binding assays of VAP and human proteins containingFFAT motifsPDA, glutatione S-transferase pulldown assay; Y2H, yeast two-hybrid; IP, coimmu-noprecipitation; S, strong binding; W, weak binding; ND, no data; �, the proteinbinds to VAP or localizes at ER; �, the protein does not bind to VAP or does notlocalize at ER.

PDAa Y2H IP ER localization

OSBP S �b �b �b,c

ORP1 W ND ND �d

ORP2 W ND ND �d

ORP3 W ND ND �e

ORP4 S ND ND �f

ORP6 S ND ND �e

ORP7 S ND ND �e

ORP9 S �a ND �a,c

NIR1 ND ND �g �g

NIR2 ND ND �g �g,h

NIR3 ND ND �g �g,h

CERT ND ND �i �h,i

a From Ref. 6.b From Ref. 4.c Overexpression of protein containing a FFAT motif.d From Ref. 29.e From Ref. 30.f From Ref. 31.g From Ref. 7.h Coexpression of VAP and protein containing a FFAT motif.i From Ref. 8.

Solution Structure of OSBP VAP-A Complex

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aaaaatgcattctcatcatct-3�); F360A forward (5�-gatgagaatgaatttg-ctgatgcacctgagat-3�) and reverse (5�-atctcaggtgcatcagcaaattca-ttctcatc-3�); F360Q forward (5�-tgatgagaatgaatttcaagatgcacctg-agatc-3�) and reverse (5�-gatctcaggtgcatcttgaaattcattctcatca-3�); D361A forward (5�-gaatgaattttttgctgcacctgagatca-3�) andreverse (5�-tgatctcaggtgcaccaaaaaattcattc-3�); E364K forward(5�-tttttgatgcacctaagatcatcaccatg-3�) and reverse (5�-catggtga-tgatcttaggtgcatcaaaaa-3�). To generate the E356K/E364K dou-ble mutant, E356K was initially prepared, and then the E364Kmutation was introduced into E356K. All mutant OSBPFs wereoverexpressed inE. coliRosetta (DE3) grown inLBmediumandthen purified using the same method as described above.The expression vector for the P56Smutant ofVAP-AMSPwas

prepared using QuikChange (Stratagene) using forward primer5�-cggtactgtgtgaggtccaacagtggaatta-3� and reverse primer 5�-taattccactgttggacctcacacagtaccg-3�. 15N-Labeled P56S VAP-AMSP was overexpressed in E. coli Rosetta (DE3) grown in M9medium containing 15NH4Cl and unlabeled glucose and thenpurified using the same method as described above.NMR Samples of the Complex—NMR samples of the com-

plex were prepared in 93% H2O, 7% D2O (v/v) containing 50mM potassium phosphate (pH 6.9), 100 mM KCl, 1 mM DTT,and 0.1 mM EDTA. Six complexes between VAP-AMSP andOSBPF were prepared: 13C,15N or 15N-labeled VAP-AMSP withunlabeled OSBPF, unlabeled VAP-AMSP with 13C,15N or 15N-labeled OSBPF, 13C,15N -labeled VAP-AMSP with 13C,15N-la-beled OSBPF, and 15N-labeled VAP-AMSP with 15N-labeledOSBPF. Complex formation was monitored using the 15NHSQC spectrum of themixture of VAP-AMSP andOSBPF. Peakpositions of the complex were identified by the 1H,15N HSQCspectrum of 15N-labeled VAP-AMSP or 15N-labeled OSBPF inthe presence of an excess of unlabeled OSBPF or unlabeledVAP-AMSP, respectively.NMR Experiments—All NMR experiments were performed

using a Bruker AV500 or DRX800 spectrometer at 303 K. Allspectrawere processed usingNMRpipe (33) and analyzed usingSPARKY (T. D. Goddard and D. G. Kneller, University of Cali-fornia, San Francisco). NMR spectra used for resonance assign-ments of the complex were as previously reported (32). Forbackbone resonance assignments of free VAP-AMSP and freeOSBPF, HNCACB, HN(CO)CACB, HN(CA)CO, and HNCOspectra (34, 35) of 13C,15N-labeled freeVAP-AMSP and 13C,15N-labeled free OSBPF were recorded, respectively. To obtain thedistance restraints of the complex, the three-dimensional 13C-edited NOESY spectrum of 13C,15N-labeled complex, three-dimensional 15N-edited NOESY, and two-dimensional 15N-filtered 1H,1H NOESY spectra of 15N-labeled complex and13C-edited (F2)/13C-filtered (F1) NOESY spectrum of the com-plex between 13C,15N-labeledVAP-AMSP and unlabeledOSBPF(34, 35) were recorded. To obtain �1 angles, HNHB andHN(CO)HB spectra (36, 37) and three bond JC�C� and JNC�

couplings (38) were measured.The 15N longitudinal spin-relaxation rates (R1) were mea-

sured with relaxation delays of 16.5*, 38.5, 60.5*, 115.5, 203.5,335.5, 555.5, and 885.5 ms (35). The 15N transverse relaxationrates (R2) were measured with relaxation delays of 16*, 32, 48*,64, 96, 112, and 128 ms (35). In these measurements, timepoints marked with an asterisk were duplicated for error esti-

mations. 1H,15N hetero-NOEs were recorded with and withoutproton saturation in an interleaved fashion (35).Titration experiments of 15N-labeled VAP-AMSP with unla-

beledOSBPF (WT)were performed in 90%H2O, 10%D2O (v/v)containing 50 mM potassium phosphate (pH 6.9), 100 mM KCl,1 mM DTT, and 0.1 mM EDTA and in 90% H2O, 10% D2O (v/v)containing 50 mM potassium phosphate (pH 6.9), 500 mM KCl,1 mM DTT, and 0.1 mM EDTA. Titration experiments of 15N-labeled VAP-AMSP with unlabeled OSBPF (E356K) was per-formed in 90% H2O, 10% D2O (v/v) containing 50 mM potas-sium phosphate (pH 6.9), 100 mM KCl, 1 mM DTT, and 0.1 mM

EDTA. For all titration experiments, the initial concentration ofVAP-AMSP was set to 0.17 mM. For the titration with OSBPF(WT), 3.17 mMOSBPF (WT) was added to VAP-AMSP at molarratios to VAP-AMSP of 0.15, 0.30, 0.44, 0.59, 0.73, 0.88, 1.02,1.17, 1.46, and 1.76. For the titration with OSBPF (E356K), 2.30mMOSBPF (E356K) was added to VAP-AMSP with molar ratiosto VAP-AMSP of 0.15, 0.30, 0.59, 0.88, 1.17, 1.46, 1.76, 2.34, 3.51,and 5.85. Finally, at 500 mM KCl, 1.69 mM OSBPF (WT) wasadded to VAP-AMSP with molar ratios to VAP-AMSP of 0.15,0.30, 0.44, 0.59, 0.73, 0.88, 1.02, 1.17, 1.46, 2.34, 3.51, 5.85, and10.5. For the line-shape analysis, the LineShapeKin Simulation4.1.1 software (39) was used with the following parameters;Ka � 2.8 � 105 M�1, koff � 20, 40, 80, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 700, 1000, and 2000 s�1, fre-quency for 15N of Glu-91 � 40,748 and 41,237 s�1 for free andcomplex, respectively, R2� 11 and 17 s�1 for free and complex,respectively, receptor concentration � 0.16–0.17 mM, and theligand/receptor ratio � 0.15, 0.30, 0.44, 0.59, 0.73, 0.88, 1.02,1.17, 1.46, and 1.76. The dilution effects were corrected usingthe LineShapeKin software for the peaks with non-linearbehavior.

15N Relaxation Analysis—All R1, R2, and 1H,15N hetero-NOE values were measured using the AV500 spectrometer at303 K. Peak height intensities in each spectrumwere measuredusing SPARKY. 1H,15N hetero-NOE values were obtained bycalculating the ratios of the peak height intensities in the spec-tra with and without 1H saturation. For determination of theoverall rotational correlation time (�C) of the complex, R1 andR2 values were obtained using the Sparky2rate software (LoriaLab), which employs a relaxation peak intensity file generatedby Sparky and fits the rate constants using the nonlinear least-squares fitting programCURVEFIT (40). Then, �C for the com-plex was calculated using the program TENSOR 2.0 (41)assuming isotropic, axial symmetric, and fully anisotropicmotion, and the isotropic model was selected. For determina-tion of the residue-specific order parameters (S2) of VAP-AMSPin the bound and unbound states, the exponential decay curvesof R1 and R2 were fitted to a two-parameter exponential equa-tion using in-house developed software. The uncertainties ofthe relaxation rates were estimated by 500 Monte-Carlo simu-lations using the intensity deviations of duplicated time points.Base-line noise levels were used for the uncertainties in hetero-NOE experiments. Rotational diffusion tensors and Lipari-Szabo model-free analyses for VAP-AMSP in the unbound andbound states were achieved using TENSOR 2.0 (41). Only res-idues having values higher than 0.7 in the hetero-NOE datawere considered for determination of the rotational diffusion

Solution Structure of OSBP VAP-A Complex

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tensor. Anisotropic and isotropic rotational diffusion tensorswere assumed for the unbound and bound states of VAP-AMSP,respectively. Using these tensors, S2 were calculated.Structure Calculation—The NOE cross-peaks in the three-

dimensional 13C-editedNOESY, three-dimensional 15N-editedNOESY, and two-dimensional 1H,1H NOESY spectra wereassigned using the CANDID algorithm of CYANA (42). A totalof 2796 upper distance restraints were obtained by CANDID.The NOE cross-peaks in the three-dimensional 13C-edited(F2)/13C-filtered (F1) NOESY spectrum were assigned manu-ally, and 29 intermolecular upper distance limits were obtained.All other intermolecular NOEswere assigned throughCANDID/CYANA cycles and confirmed by visual inspections. 141backbone torsion angle restraints were derived using theTALOS program (43), 10 �1 angle restraints were obtainedfrom HNHB and HN(CO)HB spectra, and 3 �1 angle restraintswere obtained by three bond JC�C� and JNC� couplings. Withthese restraints, a total of 100 structures that did not show sig-nificant violations were generated by 20,000 time-step dynam-ics using CYANA (44, 45), and these were further refined byAMBER 9 (46) using an all-atom force field (ff99SB). TheAMBER refinement consists of three stages: 1500 steps ofenergy minimization, 20-ps molecular dynamics, and 1500steps of energy minimization. To take into account the electro-static and solvent-solute interactions precisely, a cut-off valuefor the non-bonded interactionwas set to infinity (cut� 999.0),and a generalized Born model (igb � 5 and gbsa � 1) (47) wasused. The best 20 structures were selected and analyzed usingAQUA and PROCHECK-NMR software (48). A structure clos-est to the lowest energy was employed as being representative.ITC—Before the ITC experiments, purified protein and pep-

tides were dialyzed against 50 mM potassium phosphate buffer(pH 6.9) containing 100 mM KCl, 1 mM DTT, and 0.1 mM

EDTA. This dialysis buffer was used to measure heats of dilu-tion. ITC experiments were performed at 293 K on aVP-ITC oran Omega Micro Calorimeter (Microcal Inc.). The experimen-tal data were analyzed by employing the Origin-ITC softwarepackage. Heats of dilution were subtracted from the raw databefore analysis.The concentrations ofOSBPF (WTandmutants) were deter-

mined bymeasuring the absorbance at 280 nm, although that ofVAP-AMSP was not determined accurately due to trace con-taminationwith other proteins. From the ITC data analysis, theapparent stoichiometry was 1:1.17 for OSBPF:VAP-AMSP.Here, we have assumed that this difference (17%) is derivedfrom an overestimation of the VAP-AMSP concentration due tocontamination, and therefore, the concentration of VAP-AMSPwas re-calibrated for all experiments.DSC—Before the DSC experiments, WT and P56S VAP-

AMSP were dialyzed against 50 mM potassium phosphate buffer(pH 6.9) containing 100mMKCl, 1mMDTT, and 0.1mMEDTAand against 50 mM potassium phosphate buffer (pH 6.9) con-taining 100 mMKCl and 1mMDTT, respectively. The dialysatebuffer was used as a reference solution for the DSC scan. Bothcalorimetric scans were performed using nanoDSC (Calorime-try SciencesCorp.).WT (1.5mg/ml) andP56S (1.8mg/ml)werescanned from 273 to 353 K and from 273 to 343 K, respectively,with a heating rate of 1 K/min.

RESULTS

Chemical Shift Assignments—All resonances of backbonenuclei (1HN, 15N, 13C�, and 13C�) of the complex betweenVAP-AMSP and OSBPF were assigned, and more than 90% of side-chain 1H and 13C resonances of the complexwere also assigned.Details of the resonance assignments of the complex (BiologicalMagnetic Resonance Bank ID 7025) have been previouslydescribed (32). Resonances of all backbone nuclei of free VAP-AMSP and 97.6% of backbone nuclei of free OSBPF were alsoassigned using conventional triple-resonance techniques (35).Stoichiometry between VAP-AMSP and OSBPF—Initially, the

ITC experiment for the interaction of OSBPF and VAP-AMSPwas performed (supplemental Fig. S1). The result indicated thatthe molar ratio of the complex is 1:1. The apparent molecularmass of the complex was then determined by NMR relaxationspectroscopy. From the R1, R2, and 1H,15N hetero-NOE values,the �c value of the complex at 303 K was determined to be12.6 � 0.1 ns using TENSOR 2.0. Because the �c value of aglobular protein is roughly proportional to the apparentmolec-ular mass, a linear correlation plot between �c and apparentmolecular mass values was obtained as shown in supple-mental Fig. S2. According to the obtained plot (supplementalFig. S2), the �c value of 12.6 ns indicates that the apparent molec-ularmass value is between 19.6 and 32.2 kDa (95% accuracy). Themolecularmass of the complex comprising of oneVAP-AMSP andone OSBPF is 20,249.7 Da. Therefore, the apparent molecularmass value of the complex together with the ITC study indicatesthat the complex consists of one VAP-AMSP and one OSBPF.Structure Determination of the Complex of VAP-A MSP

Domain with OSBP Fragment—The solution structure of thecomplex of VAP-AMSP with OSBPF was determined. For struc-

TABLE 2Structural statistics for the complex between VAP-AMSP and OSBPF

NOE upper distance restraintsIntramolecularIntraresidual (�i � j� � 0) 691Sequential (�i � j� � 1) 824Medium range (1 � �i � j� � 5) 365Long range (��i � j�) 858

Total 2742Intermolecular 83(29a)

Dihedral angle restraints� 69� 68�1 13

AMBER GB energies (kcal/mol)Total �5825 � 6Constraints 15 � 1

Maximum violationsDistance (Å) 0.216Angle (°) 4.868

Mean deviations from ideal geometryBond lengths (Å) 0.0098 � 0.0001Bond angles (°) 2.29 � 0.02

RMSD (VAP-A 6–125, OSBP 358–366) (Å)Backbone atoms 0.66 � 0.14Heavy atoms 1.19 � 0.16

Ramachandran analysis (VAP-A 6–125,OSBP 358–366) (%)

Most favored regions 90.6Additional allowed region 8.1Generously allowed regions 1.4Disallowed regions 0.0

a Distance restraints obtained from three-dimensional 13C-edited (F2)/13C-filtered(F1) NOESY.

Solution Structure of OSBP VAP-A Complex

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ture calculations of the complex, the NOE cross-peaksobserved in most NOESY spectra were assigned using theCANDID algorithm of CYANA, although the intermolecularNOEcross-peaks observed in the three-dimensional 13C-edited(F2)/13C-filtered (F1)NOESY spectrumwere assignedmanually(supplemental Fig. S3). A total of 2825 distance restraints wereobtained, and 29 of 83 intermolecular NOEs were derived fromthe three-dimensional 13C-edited (F2)/13C-filtered (F1) NOESYspectrum (Table 2). The other restraints for structure calcula-tions are shown in Table 2. Using these restraints, structures ofthe complex were calculated. A superimposed representation

of the final 20 lowest energy struc-tures obtained is shown in Fig. 2A.The root mean square deviations ofbackbone and heavy atoms over resi-dues 6–125 of VAP-AMSP and resi-dues 358–366 of OSBPF were 0.66and 1.19 Å, respectively (Table 2).Other structural statistics are pro-vided in Table 2.Global Structure—VAP-AMSP has

an immunoglobulin-like �-sandwichfold consisting of seven �-strandsand one�-helix (Fig. 2B). The struc-ture of VAP-AMSP is similar to MSP(PDB ID 1MSP), and the positionalroot mean square deviation ofsuperimposed C� atoms is 2.0 Å.The FFAT motif of OSBPF adoptsan extended �-strand-like confor-mation and is bent at theC terminusof the FFAT motif (Fig. 2, A and B).OSBPF has an overall negativelycharged surface and binds to VAP-AMSP at a positively charged surface(Fig. 2C).Interaction between VAP-A and

OSBP Fragment—Details of theintermolecular interactions were

analyzed using MONSTER (49), and interactions observed inmore than 12 structures of the final 20 structures were adopted.The FFAT motif and C-terminal side of OSBPF interact withVAP-AMSP � strands C, D, E, and F and a loop connecting�-strands C and D. The complex of VAP-AMSP and OSBPF isstabilized by electrostatic, hydrophobic, and hydrogen-bondinteractions. Fig. 3 shows the interaction site of VAP-AMSP andOSBPF. The carboxyl groups of Asp-361 andGlu-364 of OSBPFinteract electrostatically with the side chains of VAP-AMSP Lys-43/Lys-45 and Arg-55, respectively (supplemental Fig. S4).These electrostatic interactions were well defined by 13 inter-molecularNOEs observed between the electrostatic interactionpair and its neighboring residues; that is, 5 NOEs betweenThr-46 and Ala-362, 2 NOEs between Lys-45 and Ala-362,Thr-46 and Phe-360, and Val-54 and Glu-364 and 1 NOEbetween Lys-45 and Asp-361 and between Val-44 and Ala-362(supplemental Fig. S3). The proton resonances of VAP-AMSPLys-43 � and positions showed unusual upfield shifts. Theseshifts may reflect the electrostatic interaction between OSBPFAsp-361 and VAP-AMSP Lys-43. The side chain of Phe-359 ofOSBPF binds in a hydrophobic pocket formed by residues Lys-45, Thr-47, Lys-87, and Met-89 of VAP-AMSP, and the sidechain of Ala-362 of OSBPF binds in a hydrophobic pocketformed by residuesVal-44, Thr-46, Val-54, andAsn-57 ofVAP-AMSP. The side chain of Phe-360 of OSBPF interacts hydropho-bically with Pro-49 of VAP-AMSP. The side chains of Pro-363,Glu-364, Ile-365, and Ile-366 of OSBPF also interact hydropho-bically with VAP-AMSP. Three intermolecular backbone-back-bone hydrogen bonds are observed between VAP-AMSP andOSBPF, where the amide nitrogens of Thr-46 and Val-54 of

FIGURE 2. Solution structure of the complex between VAP-A (6 –125) and OSBP (358 –366). A, shown is asuperimposed representation of 20 lowest energy structures. B, shown is a ribbon representation of VAP-AMSP.OSBPF is shown as a stick model. C, shown are the electrostatic surfaces of VAP-AMSP (left) and OSBPF (right) con-toured from �3 kT (red) to �3 kT (blue). The electrostatic potential was calculated using an amber force field.

FIGURE 3. Details of the interaction. Residue numbers of OSBPF are writtenin italics. OSBPF is shown as a stick model. The surface of VAP-AMSP is repre-sented with acidic (red), basic (blue), and hydrophobic (yellow) residues.

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VAP-AMSP and Phe-360 of OSBPF form hydrogen bonds withthe carbonyl oxygens of Phe-360 and Pro-362 of OSBPF andThr-46 of VAP-AMSP domain, respectively. Amide proton res-onances of Thr-46 and Val-54 of VAP-AMSP and Phe-360 ofOSBPF shifted downfield upon complex formation, indicatingformation of the respective hydrogen bonds.Intermolecular interactions observed in the structure were

further investigated by mutational studies, and a series ofOSBPF mutants was generated to achieve this end. For thequantitative analysis, ITC experiments were performed usingOSBPF mutants with VAP-AMSP. The dissociation constantsobtained from the ITC experiments are summarized in Table 3,and other thermodynamic parameters obtained from the ITCexperiments are summarized in supplemental Table S1. TheE356K/E364K double mutant showed the most significant lossof binding to VAP-AMSP. The F360A, D361A, and E364Kmutants also showed significant loss of binding, although the

F360Q mutant showed moderate loss of binding, and theE356K and E358A mutants showed only slight loss of binding.Disorder Property of Free OSBP—The disorder property of

OSBP was examined using the IUPred algorithm (50, 51). Theextensive region of OSBP (Q295-R406), including the FFATmotif (E358-E364), was estimated to be disordered. For exper-imental investigation of the disordered property of OSBP,1H,15Nhetero-NOEwas applied toOSBPF. This 1H,15Nhetero-NOE value is a good indicator of local environment dynamics;negative and large positive NOE values indicate flexible andrigid regions, respectively. All residues of OSBPF showed nega-tive NOE values in the unbound state (Fig. 4A), suggesting thatfree OSBPF is flexible. Structure analysis of free OSBPF usingthe TALOS program (43) with the assigned 1HN, 15N, 13C�, C�,and 13C� chemical shifts also showed no ordered structure.

Proteins containing native, unfolded functional regions(30–40 residues) under physiological conditions are referredto as intrinsically disordered proteins (52). Larger interactionsurfaces and structural plasticity in the native state is thoughtto be advantageous for intrinsically disordered proteins inrecognizing various targets with sufficient specificity butwithout huge loss of affinity. A region of more than 100consecutive residues in OSBP, including 33 residues charac-terized in this study, seem not to adopt an ordered structure.Thus OSBP belongs to the intrinsically disordered proteinfamily.Changes in Dynamics and Structure upon Complex Forma-

tion—1H,15N hetero-NOE values for the backbone amides ofOSBPF in the bound state are shown in Fig. 4A. Upon binding to

VAP-AMSP, 1H,15N hetero-NOE val-ues of the residues around the FFATmotif of OSBPF changed from nega-tive to positive, indicating thatOSBPFformed a stable structure upon bind-ing to VAP-AMSP. Fig. 4B shows thechange in S2 for the backbone amidesof VAP-AMSP upon complex forma-tion. For Tyr-46, Tyr-47, and Arg-51,which are localized at a loop regioninvolved in the binding to OSBPF, S2values increased by about 0.35 uponcomplex formation, indicating thatthe psns motion of these residueswas restricteduponbinding toOSBPFand contributed to formation of a sta-ble complex structure.Fig. 4C shows backbone amide

chemical shift changes of OSBPFand VAP-AMSP upon complexformation. Perturbed residues ofOSBPF were observed for the FFATmotif and the C-terminal side of theFFAT motif, which interact withVAP-AMSP in our structure. Per-turbed residues of VAP-AMSP wereprimarily located within or near thebinding site. Two free VAP-AMSPstructures have been previously

FIGURE 4. A, backbone 1H,15N hetero-NOE values of OSBPF in the unbound (white bar) and bound (black bar)state are shown. Uncertainties were obtained using Monte Carlo simulations. B, shown are changes of S2 valuesof VAP-AMSP upon complex formation with OSBPF. �S2 � S2(complex) � S2(free). The region between �S2 ��0.1 0.1 is shadowed. C, composite chemical shift changes of VAP-AMSP and OSBPF upon complex formationare shown. Composite chemical shift changes were calculated using the equation �ppm � {(��H)2 � (��N/5)2}1/2, where ��H and ��N represent the chemical shift changes of 1H and 15N, respectively.

TABLE 3The dissociation constants (Kd) obtained from the ITC experimentsVAP-AMSP (0.9 mM for E356K, 1.2 mM for E364K, and 1.0 mM for the others) wastitrated into OSBPF (63–70 M) at 293 K.

OSBP Kd

M

E356K 6.9 � 0.7 � 10�6

E358A 4.1 � 0.1 � 10�6

F360A 2.1 � 0.1 � 10�5

F360Q 1.1 � 0.1 � 10�5

D361A 2.8 � 0.1 � 10�5

E364K 3.5 � 0.3 � 10�5

E356K/E364K 1.1 � 0.1 � 10�4

WT 2.1 � 0.1 � 10�6

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determined (PDB IDs 2CRI3 and 1Z9L (21)). Positional rootmean square deviations of superimposedC� atoms of our struc-ture with these free VAP-AMSP structures were both 1.3 Å,except for the loop regions (Phe-76—Lys-85). Judging from thechemical shift perturbation and the similarities between ourstructure and the free structures, the structure of VAP-AMSPdoes not change markedly upon complex formation.

DISCUSSION

Comparison with the Crystal Structure—A crystal structureof a complex between the rat VAP-AMSP and the rat ORP1

fragment containing the FFATmotif (SEDEFYDALS) (ORP1F) hasbeen determined by Kaiser et al.(21). The overall structures of ourstructure and the crystal structure(PDB ID 1Z9O) are similar, and thepositional root mean square devia-tion of superimposed C� atoms ofVAP-AMSP and the FFAT motif are1.2 Å, although the crystal structureis composed of two VAP-AMSP andtwo ORP1F molecules. Clear differ-ences were observed for the intermo-lecular interactions in that side chainsof the third Phe/Tyr and fourth Aspresidues of the FFAT motif (the con-sensus sequence EFFDAXE) interactwithVAP-AMSP in the solution struc-ture (supplemental Fig. S5). In thecrystal structure, these residues donot interact with VAP-AMSP butinteract with the second ORP1F mol-ecule to stabilize the 2:2 complex (21).Substitution of the fourth Asp of theFFAT motif with Ala disrupts ERlocalization of Osh1, a yeast proteincontaining the FFAT motif (5), anddisrupts the interaction of CERT, aceramide transport protein contain-ing the FFAT motif, with VAP-A invivo (8). These results are consistentwith our solution structure as the sidechain of the fourth Asp residue of theFFAT motif interacts directly withVAP-AMSP. Thus, we assume thatformation of the solution structure,comprising a 1:1 complex betweenVAP-AMSP and a FFAT protein, isessential for bindingof the FFATpro-tein to VAP-A.Recognition Mechanism of In-

trinsically Disordered OSBPF byVAP-AMSP—In an effort to furtherunderstand the recognition mecha-nism of OSBPF by VAP-AMSP, we

inspected the perturbed chemical shift patterns in titrationexperiments that were carried out using 15N-labeled VAP-AMSP or OSBPF. Nonlinear behavior was found for eight resi-dues of VAP-AMSP, whereas the other peaks show fast/interme-diate line shapes with linear patterns (supplemental Fig. S6).The line shape simulation using the LineShapeKin softwarepackage could only account for the behavior of four residues,although the other four residues, Ser-58, Gln-74, Gln-91, andCys-121, remained elusive using the two-site exchange model(supplemental Fig. S6). The complicated features of these resi-dues were revealed by inspecting the line shapes of these resi-dues. For instance, Cys-121 consisted of 4 peaks at a 1:0.44molar ratio of VAP-AMSP to OSBPF as shown in Fig. 5A. The3 H. Endo, F. Hayashi, M. Yoshida, and S. Yokoyama, unpublished information.

FIGURE 5. A, shown are cross-peaks of Cys-121 at a molar ratio of OSBPF to VAP-AMSP of 1:0. 44. In the upperpanel, peaks are indicated by a cross and are labeled. The lower panel shows 1H cross-sections corresponding tolines a, b, and c in the upper panel. Numbers indicate corresponding peaks in the upper panel. B, shown is anoverlay of 15N cross-sections of Gln-91 in a titration of VAP-AMSP with OSBPF. Molar ratios (VAP-AMSP:OSBPF)ranged from 1:0 (red) to 1:1.76 (blue). Peak tops at a molar ratio of 1:0.59 are shown by arrows. C, the upper panelshows an overlay of cross-peaks for Gln-74 at molar ratios of OSBPF of 0 (red), 0.59 (purple), and 1.76 (blue). Thelower panel shows 1H cross-sections of peaks at each molar ratio. Corresponding peaks in the upper and lowerpanels are connected by dashed lines. D, shown is a comparison of cross-peaks for Cys-121 in titrations withOSBPF (WT) and the E356K mutant.

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overlaid one-dimensional titration curve of Gln-91 also dis-played 3 peak tops at a molar ratio of 1:0.59 (Fig. 5B). Glu-74showed two peaks at a molar ratio of 1:0.59 (Fig. 5C). One isfound at 8.03 ppm 1H between free (8.13 ppm) and complex(7.99 ppm) peak positions, and the other (8.14 ppm) is out ofrange. A convincing explanation for these observations is toassume formation of intermediate states. The intermediatecomplex is thought to be maintained by nonspecific interac-tions considering the observations that the peaks in the middleof the titration have several peak tops and there is no clearintermolecular NOE.We hypothesized that electrostatic interactions between

OSBPF and VAP-AMSP play a role in forming the intermediate,as initial screening of sample conditions showed that the titra-tion under 500mMKCl does not reach saturation even at a 10:1ratio of OSBPF to VAP-AMSP (data not shown). Additionally,VAP-AMSP has a large basic area at the same surface in theregion, whichOSBPF binds to (Fig. 2C), and a plethora of acidicresidues are found in OSBPF as well as its homologoussequences. In addition to the three acidic residues that arewithin the FFAT motif, there is an acidic patch of 5 residues

comprising Asp-352, Glu-353, Asp-354, Asp-355, and Glu-356 at aregion preceding the FFAT motif.This acidic patch region does notconverge in the final ensemblestructures due to the absence ofmedium to long range intra- andintermolecular NOE restraints, sug-gesting that there is no stable inter-action between the acidic patch andVAP-AMSP. This is also indicated bysmall differences in chemical shiftsbefore and after binding to VAP-AMSP and the lower hetero-NOEvalues of these residues in the com-plex (Fig. 4, A and B). We examinedthe role of the acidic patch by pre-paring the charge reversal E356Kmutant of OSBPF and conductingtitration experiments using 15N-la-beled VAP-AMSP. Remarkably inthis case, with the exception ofGln-74 and Gln-91, the nonlinearpatterns that were found in titra-tions withWT disappeared, therebyallowing for simulation using a two-site model (Fig. 5D and supple-mental Fig. S7). It should be notedthat the chemical shifts of VAP-AMSP in the saturated state are sim-ilar even in the complex with theE356K mutant, implying that thefinal structures are similar. None-theless, the ITC experiment showedthat the E356K mutant has reducedbinding affinity for VAP-AMSP(Table 3). In a three-site exchange

consisting of free, intermediate, and binding complex, theexchange between the intermediate and final product isexpected to bemuch faster than that between the free and inter-mediate states. The E356K mutant probably possesses anincreased off-rate from the intermediate to free conformation,which in turn leads to higher values of Kd and a faster apparentexchange rate. These results suggest the possibility that theacidic patch of OSBPF, including Glu-356 of OSBPF, takes partin increasing the apparent binding affinity by contributing tothe formation of an intermediate complex with VAP-AMSPthrough electrostatic interactions.Effects of P56SMutation on VAPMSP—The P56S mutation in

human VAP-B causes familial autosomal dominant motoneu-ronal diseases, ALS8, which exhibits a typical ALS phenotypewith rapid progression or late onset spinal muscular atrophy(22). The P56S mutation in VAP-B induces insolubility andaggregate formation of VAP-B (23). Pro-56 is conserved notonly in the VAP family of proteins but also in theMSP family ofproteins (53). To investigate the effects of the P56Smutation onVAPMSP, we prepared the P56S mutant of VAP-AMSP. Fig. 6Ashows the 1H,15N HSQC spectrum of P56S VAP-AMSP super-

FIGURE 6. A, 1H,15N HSQC spectra of P56S (red) and WT (blue) VAP-AMSP are shown. In the boxed region, assign-ments of peaks of WT VAP-AMSP are indicated. B, 1H,15N HSQC spectra of P56S VAP-AMSP in the presence ofOSBPF with a molar ratio of 1:2 (red) and in the absence of OSBPF (blue) are shown. C, DSC profiles of P56S (red)and WT (black) VAP-AMSP are shown.

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imposed on that ofWTVAP-AMSP. The number of peaks in the1H,15N HSQC spectrum of P56S VAP-AMSP was greater thanthat expected from the amino acid sequence of P56S VAP-AMSP. Some residues of P56S VAP-AMSP probably gave doublepeaks, and the intensities of these double peaks were similarwithout obvious line-broadenings. These residues did not mapto a specific region, although the assignments were not com-pleted. Hence, it is concluded that P56S VAP-AMSP has twoconformations with similar abundance.We then investigated the ability of P56S VAP-AMSP to bind

the FFAT motif. Fig. 6B shows the 1H,15N HSQC spectrum ofP56S VAP-AMSP in the presence of OSBPF superimposed onthat in the absence of OSBPF. Double peaks in both were per-turbed by the addition of OSBPF. Therefore, both conforma-tions of P56S VAP-AMSP can bind to OSBPF. In the presence ofOSBPF, some double peaks have similar intensity (Fig. 6B,upper inset), whereas others have different intensities (Fig. 6B,lower inset). It seems that the binding modes for OSBPF differbetween the two conformations.We then investigated the thermodynamic stability of P56S

VAP-AMSP by DSC (Fig. 6C). The temperature of maximumheat capacity of WT VAP-AMSP is 64.6 °C and that of P56SVAP-AMSP is 49.0 °C, indicating that P56S VAP-AMSP is ther-mally unstable. The heat capacity peak of P56S VAP-AMSP isbroader than that ofWT, indicating that the change in enthalpy(�H) of protein unfolding of P56S VAP-AMSP is smaller thanthat of WT. In fact, DSC curve analyses showed that �H ofprotein unfolding of WT and P56S VAP-AMSP is 529 and 416(436 at 64.6 °C) kJ/mol, respectively. The decrease in �H ofprotein unfolding associated with the P56S mutation indicatesthat the thermal instability of P56S VAP-AMSP results fromlosses of hydrogen bonds and/or van der Waals interactions inthe protein. As judged by the 1H,15NHSQC spectra of P56S andWTVAP-AMSP, the structure of P56S VAP-AMSP does not dif-fer greatly from that ofWT.However, results of theDSC exper-iments showed that P56S VAP-AMSP is more thermally unsta-ble thanWT. Because the MSP domains of VAP-A and VAP-Bshare 82% amino acid sequence identity, their structures areprobably similar. It is likely that the effects of the P56Smutationon VAP-BMSP are similar to the effects of the P56Smutation onVAP-AMSP. Thus, it is reasonable to assume that the P56Smutation in VAP-B decreases the thermal stability of VAP-Band facilitates aggregation.Concluding Remarks—In this report, we have determined the

solution structure of the complex between VAP-AMSP andOSBPF and found thatmost residues in the FFATmotif interactwith VAP-AMSP. This solution structure explained the roles offive of six conserved residues in the FFAT motif, and three offive were not explained before. Furthermore, we found that 1)VAP-AMSP andOSBPF form a complex with amolar ratio of 1:1in solution, 2) the central region of OSBP including the FFATmotif is intrinsically disordered, 3) VAP-AMSP andOSBPF forman intermediate complex before forming a stable 1:1 complex,4) electrostatic interactions are important for binding, and 5) atleast one acidic residue in an acidic patch preceding the FFATmotif is involved in intermediate complex formation andenhances the binding. These findings suggest that disorderedOSBP initially binds VAP-A through nonspecific charge inter-

actions involving acidic residues at the N-terminal side of theFFAT motif and finally forms a stable complex structurethrough a “fly-casting”-like process (54, 55).

Acknowledgments—We thank M. Yoneyama, H. Kinoshita, and A.Yashuba for technical assistance.

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Kyoko Furuita, JunGoo Jee, Harumi Fukada, Masaki Mishima and Chojiro KojimaProtein A Revealed by NMR and Mutagenesis Studies

Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated

doi: 10.1074/jbc.M109.082602 originally published online February 23, 20102010, 285:12961-12970.J. Biol. Chem. 

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  http://www.jbc.org/content/suppl/2010/02/23/M109.082602.DC1

  http://www.jbc.org/content/285/17/12961.full.html#ref-list-1

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